High-Performance Liquid Chromatographic Determination of Cholesterol and Cholestanol in Human Serum by Precolumn Derivatization with 2-(2-(Isocyanate)ethyl)-3-methyl-1,4-naphthoquinone Combined with Platinum Catalyst Reduction and Electrochemical Detection.

Abstract

The precolumn derivatization reagent, 2-[2-(isocyanate)ethyl]-3-methyl- 1,4-naphthoquinone (IMQ) was applied to the simultaneous determination of cholesterol and cholestanol in human serum by HPLC with electrochemical detection. The compounds extracted with hexane from human serum, to which was added 1-eicosanol as an internal standard, were reacted with IMQ in acetone and converted into the corresponding carbamic acid esters. After chromatographic separation of the derivatives, they were reduced once in a platinum catalyst reduction column connected on-line, then quantified with an electrochemical detector in the oxidation mode. The detection limits at a signal-to-noise ratio of 3 were 6.6 pg (17 fmol) and 7.4 pg (19 fmol) for cholesterol and cholestanol, respectively in a 10 microliter injection volume. This method was successfully applied to the determination of free and total cholesterol and total cholestanol in normal human serum, and the concentrations of free and total cholesterol were compared with those obtained by an enzymatic method.